Spherical nucleic acid-based constructs as immunostimulatory agents for prophylactic and therapeutic use

ABSTRACT

Aspects of the invention relate to spherical nucleic acid-based constructs and related methods and compositions thereof. The compositions of the invention are useful for activating agonists of nucleic acid interacting complexes, such as TLRs, stimulating an immune response, and treating diseases such as infectious disease, cancer, allergies, allergic diseases, and autoimmune disease

RELATED APPLICATIONS

This Application is a continuation of U.S. application Ser. No.14/907,430, filed Jan. 25, 2016, entitled “SPHERICAL NUCLEIC ACID-BASEDCONSTRUCTS AS IMMUNOSTIMULATORY AGENTS FOR PROPHYLACTIC AND THERAPEUTICUSE”, which is a national stage filing under 35 U.S.C. 371 ofInternational Patent Application Serial No. PCT/US2014/048291, filedJul. 25, 2014, entitled “SPHERICAL NUCLEIC ACID-BASED CONSTRUCTS ASIMMUNOSTIMULATORY AGENTS FOR PROPHYLACTIC AND THERAPEUTIC USE”, which isa Non-Prov of Prov (35 USC 119(e)) of U.S. application Ser. No.61/858,558, filed Jul. 25, 2013, entitled “SPHERICAL NUCLEIC ACID-BASEDCONSTRUCTS AS IMMUNOSTIMULATORY AGENTS FOR PROPHYLACTIC AND THERAPEUTICUSE”. The entire contents of these applications are incorporated hereinby reference in their entirety.

FIELD OF INVENTION

The invention relates to nanoscale constructs of agonists of nucleicacid-interacting complexes, such as agonists of TLR, as well as methodsand compositions thereof.

BACKGROUND OF INVENTION

The immune system is a highly evolved, exquisitely precise endogenousmechanism for clearing foreign, harmful, and unnecessary materialincluding pathogens and senescent or malignant host cells. It is knownthat modulating the immune system for therapeutic or prophylacticpurposes is possible by introducing compounds that modulate the activityof specific immune cells. A primary example is vaccines, which haveshown the ability to induce protection against pathogens as well ascancerous cells. The first modern vaccine formulations includedlive/attenuated or inactivated pathogens, but these were deemed tootoxic in many instances or did not provide protective immunity. Purifiedprotein derivatives and other antigenic subunit vaccine strategies havebeen pursued, but these typically lead to mildly protective orinefficient responses. It is now appreciated that effective immunity, inmost instances, is known to require use of immunostimulatory compounds,which, among other things, provide the necessary signals to induce morerobust, specific, and long-lived responses, including cell-mediatedimmunity and immunologic memory. The nature of these responses can bemodulated by the type of immunostimulatory compound(s) introduced.Indeed, it has been postulated that immunostimulatory compoundsadministered together in the presence of appropriate antigenic stimulican be used to elicit a wide variety of immune responses, with thepotential to treat or prevent various ailments, including infectiousdiseases and cancer. These can also potentially be used to vaccinateimmunocompromised populations, such as children and the elderly.¹

Existing vaccines fail to induce effective immune responses in a varietyof diseases with critical worldwide impact, including AIDS, malaria,chlamydia, various malignancies and allergies or allergic diseases, suchas asthma. Among the immunostimulatory compounds being developed,agonists of Toll-like receptors (TLR) have demonstrated outstandingpotential. Agonists of TLR4, such as monophosphoryl lipid A (MPL) havereached late stages of clinical trials and approval in various countriesin some instances.² Despite these promising results, there is still aclear and significant need for compounds which can safely and effectiveinduce responses that can clear intracellular pathogens and cancers,such as inducers of cell-mediated immunity. Agonists of TLR 3, TLR 7/8and TLR 9 have excellent potential due to their potent ability to induceThl cell-mediated immune responses. A synthetic TLR 7/8 agonist,imiquimod, has been approved to treat various skin diseases, includingsuperficial carcinomas and genital warts, and is being developed for avariety of other indications. Similarly, agonists of TLR 9 are invarious stages of clinical development, for treatment of variousdiseases with large unmet medical needs. However, concerns due to lackof efficacy, off-target phosphorothioate effects, and toxicity haveslowed effective clinical translation of TLR 7/8 and 9 agonists.

SUMMARY OF INVENTION

Described herein are novel methods and compositions for enhancing immuneresponses and activating nucleic acid interacting complexes such as TLRsusing a nanoscale construct. Aspects of the invention relate tonanoscale constructs having a corona of an agonist of nucleicacid-interacting complexes wherein the surface density of the agonist ofnucleic acid-interacting complexes is at least 0.3 pmol/cm².

In other aspects the invention is a nanoscale construct having a coronaof an agonist of nucleic acid-interacting complexes, and an antigenincorporated into the corona. In some embodiments the surface density ofthe antigen is at least 0.3 pmol/cm². In other embodiments the antigenincludes at least two different types of antigen.

In yet other aspects the invention is a nanoscale construct having acorona with at least two agonists of nucleic acid-interacting complexesincorporated, wherein the agonists are selected from the groupconsisting of TLR 3, 7/8, and/or 9 agonists.

In some embodiments the agonist of nucleic acid-interacting complexescontains a spacer.

In other embodiments the agonist of nucleic acid-interacting complexesis RNA or DNA. The agonists of nucleic acid-interacting complexes maybe, for instance, a double stranded RNA, such as poly(I:C).Alternatively the agonist of nucleic acid-interacting complexes may be asingle stranded RNA such as an RNA containing UUG-motifs. In someembodiments the agonist of nucleic acid-interacting complexes is anunmethylated deoxyribonucleic acid, such as a CpG oligonucleotide.

The nanoscale construct, in some embodiments, contains a nanoparticlecore at the center of the corona which is optionally metallic. Themetallic core may be selected from the group consisting of gold, silver,platinum, aluminum, palladium, copper, cobalt, indium, nickel andmixtures thereof. In some embodiments the nanoparticle core comprisesgold. In other embodiments the nanoscale construct is degradable.

In certain embodiments, the diameter of the nanoscale construct is from1 nm to about 250 nm in mean diameter, about 1 ran to about 240 nm inmean diameter, about 1 nm to about 230 nm in mean diameter, about 1 nmto about 220 nm in mean diameter, about 1 nm to about 210 nm in meandiameter, about 1 nm to about 200 nm in mean diameter, about 1 nm toabout 190 nm in mean diameter, about 1 nm to about 180 nm in meandiameter, about 1 nm to about 170 nm in mean diameter, about 1 nm toabout 160 nm in mean diameter, about 1 nm to about 150 nm in meandiameter, about 1 nm to about 140 nm in mean diameter, about 1 nm toabout 130 nm in mean diameter, about 1 nm to about 120 nm in meandiameter, about 1 nm to about 110 nm in mean diameter, about 1 nm toabout 100 nm in mean diameter, about 1 nm to about 90 nm in meandiameter, about 1 nm to about 80 nm in mean diameter, about 1 nm toabout 70 nm in mean diameter, about 1 nm to about 60 nm in meandiameter, about 1 nm to about 50 nm in mean diameter, about 1 nm toabout 40 nm in mean diameter, about 1 nm to about 30 nm in meandiameter, or about 1 nm to about 20 nm in mean diameter, or about 1 nmto about 10 nm in mean diameter.

In other aspects the invention is a nanoscale construct of a corona ofan agonist of nucleic acid-interacting complexes, wherein the agonist isnucleic acid having at least one phosphodiester internucleotide linkage.In some embodiments the agonist is a CpG oligonucleotide. In otherembodiments each internucleotide linkage of the nucleic acid is aphosphodiester linkage.

In embodiments of the invention the corona is a spherical corona.

A vaccine composed of a nanoscale construct described herein and acarrier is provided according to other aspects of the invention.

A method for delivering a therapeutic agent to a cell by delivering thenanoscale construct of the invention to the cell is provided in otheraspects.

A method for regulating expression of a target molecule is provided inother aspects of the invention. The method involves delivering thenanoscale construct of the invention to the cell. In some embodimentsthe target molecule is a TLR selected from the group consisting of TLR3,7, 8, and 9.

A method for activating a TLR by delivering the nanoscale construct asdescribed herein to the cell is provided in other aspects of theinvention.

According to other aspects the invention is a method of treating asubject, involving administering to the subject the nanoscale constructas described herein in an effective amount to stimulate an immuneresponse. In some embodiments the subject has an infectious disease, acancer, an autoimmune disease, allergy, or an allergic disease such asasthma.

In yet other embodiments, the invention is a method of inducing animmune response in a subject, by administering to the subject ananoscale construct of a corona of an agonist of nucleicacid-interacting complexes, wherein the agonist is nucleic acid havingat least one phosphodiester internucleotide linkage in an effectiveamount to stimulate an immune response.

In certain embodiments, the method involves delivering a therapeutic ordetection modality to a cell.

Further aspects of the invention relate to a kit comprising: a nanoscaleconstruct optionally including a nanoparticle core; and having anagonist of nucleic acid-interacting complexes and instructions forassembly of an agonist of nucleic acid-interacting complexes-corona. Incertain embodiments, the kit further comprises instructions for use.

Each of the limitations of the invention can encompass variousembodiments of the invention. It is, therefore, anticipated that each ofthe limitations of the invention involving any one element orcombinations of elements can be included in each aspect of theinvention. This invention is not limited in its application to thedetails of construction and the arrangement of components set forth inthe following description or illustrated in the drawings. The inventionis capable of other embodiments and of being practiced or of beingcarried out in various ways.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings are not intended to be drawn to scale. In thedrawings, each identical or nearly identical component that isillustrated in various figures is represented by a like numeral. Forpurposes of clarity, not every component may be labeled in everydrawing. In the drawings:

FIGS. 1A-1B show a schematic non-limiting example of a nanoscaleconstruct of the invention. FIG. 1A. A general structure of an adjuvantnanoscale construct having a core and one or more agonists of nucleicacid interacting complexes, such as TLR agonists bound thereto is shown.FIG. 1B. A general structure of an adjuvant nanoscale construct having acore and one or more TLR agonists and one or more antigens bound theretois shown.

FIG. 2 is a set of graphs depicting markedly enhanced potency inmacrophages of the nanoscale constructs of the invention over agonistsof nucleic acid-interacting complexes (CpG oligonucleotides) insolution.

FIG. 3 is a set of graphs depicting markedly enhanced potency inmacrophages of the nanoscale constructs of the invention over agonistsof nucleic acid-interacting complexes (CpG oligonucleotides) in solutionfollowing overnight incubation.

FIG. 4 is a set of graphs depicting an enhanced level of cytokinesecretion with the nanoscale constructs of the invention over agonistsof nucleic acid-interacting complexes (CpG oligonucleotides) insolution. The effect on cytokine induction was examined for botholigonucleotides having phosphodiester and phosphorothioateinternucleotide linkages in both the nanoscale construct and the TRLagonist groups.

FIG. 5 is a line and bar graph depicting TLR9 activation in response tostimulation with a nanoscale construct of the invention having aphosphodiester CpG oligonucleotide in comparison with phosphodiester andphosphorothioate CpG oligonucleotides in solution.

FIG. 6 is a set of graphs depicting multiple fold increase in potency ofa nanoscale construct of the invention over several different CpGoligonucleotide sequences. CpG 1826 is SEQ ID NO: 1 and the rp1V belowit is SEQ ID NO: 2. CpG 1668 is SEQ ID NO: 3 and the rp1V below it isSEQ ID NO: 4.

FIGS. 7A-7B are a set of graphs depicting the effects of modulatingnanoscale construct core size suggests on the enhancement of agonistactivity. FIG. 8 shows a graph depicting more rapid and sustainedactivation than CpG oligonucleotide.

FIG. 9 is a set of graphs depicting the ability of phosphorothioatemodifications to modulate agonist activity in a sequence-dependentmanner. For CpG 1668: PO is SEQ ID NO: 5, C*G is SEQ ID NO: 6, 5PS2/C*Gis SEQ ID NO: 7, and PS is SEQ ID NO: 8. For CpG 1826: PO is SEQ ID NO:9, C*G is SEQ ID NO: 10, 5PS2/C*G is SEQ ID NO: 11, and PS is SEQ ID NO:12.

FIG. 10 is a set of graphs depicting the ability of oligonucleotideloading density to affect agonist activity.

FIG. 11 shows a graph depicting a time course of activation of CpG PO/POnanoscale constructs. The tested constructs are not activated until >4hr of incubation.

FIG. 12 shows a graph demonstrating that 5′Chol CpG PO nanoscaleconstructs show activation in low nM range, and 5′C18 abrogated theactivity.

FIG. 13 is a set of graphs demonstrating that pre-plated macrophages aremore primed for subsequent activation.

FIG. 14 is a set of graphs demonstrating low levels of IFN-gammasecretion by macrophages.

FIG. 15 shows a representation of an immunotherapeutic SNA (AST-008).FIG. 15 shows that the SNAs can co-present a therapeutic vaccine antigenand adjuvant on a single nanoparticle, and may simultaneously targetmultiple immunostimulatory receptors (e.g. TLR 3, 4, 7/8, 9).

FIG. 16 is a schematic demonstrating how AST-008 can enter endosomes viatriggered endocytosis, where it then can be used for versatile immunesystem stimulation. Within the endosome, AST-008 stimulates immunesystem signaling via the TLR 9 receptor, a molecular target for SNAtherapy, leading to both innate and adaptive immune responses. AST-008may also target TLR 3, 4, 7/8, resulting in innate and adaptive immuneresponses.

FIGS. 17A-17B are a set of graphs showing that AST-008 induces higherpro-inflammatory responses than corresponding CpG oligodeoxynucleotides(oligo) in vitro. FIG.

17A shows the expression levels of TNF, IL-12, and IL-6 induced by CTLoligo, CTL SNA, CpG 1826, and AST-008. FIG. 17B presents the NF-KBactivation stemming from the indicated agents.

FIG. 18 demonstrates that AST-008 targets draining lymph nodes afteradministration of a single subcutaneous dose. AST-008 was silver-stainedto enhance light scattering of the gold core, and then counterstainedwith eosin. 4X bright field magnification was used.

FIG. 19 is a graph illustrating the in vivo activity of AST-008. Micewere given a 50 μL bolus tail vein (intravenous) injection of 5.1 nmolsolution (AST-008-po, AST-008-ps, CpG 1826-po, CpG 1826-ps, GpC-po SNA,GpC-ps SNA, GpC-po, or GpC-ps) and then analyzed for IL-12 expression 1,3, and 6 hours after injection (24 mice per group, 3 per each timepoint). IL-12 levels are expressed as the fold over PBS. AST-008architecture enhances the induction of IL-12 by approximately 20-foldover free oligodeoxynucleotides, and the effect was sustained for oversix hours after the initial administration.

FIGS. 20A-20C consist of a pair of graphs and a chart that demonstratethat AST-008 induces both a balanced Th1/Th2 response (FIG. 20A) and ahigher IgG2a antibody (FIG. 20B) response than alum or CpGoligonucleotides. The results are tabulated in FIG. 20C. **p<0.01.

FIGS. 21A-21B show that AST-008 induces cellular responses moreeffectively than alum or CpG oligonucleotides. FIG. 21A schematicallyrepresents the protocol: splenocytes were grown for 28 days, challengedon Day 0 and Day 21, and then restimulated with SIINFEKL and probed forINF-γ with ELISPOT on Day 28. FIG. 21B is a graph depicting the results.****p<0.0001.

FIGS. 22A-22B demonstrate that AST-008 induces a profound tumor-clearingimmune response in an in vivo lymphoma model. FIG. 22A illustrates theprotocol: the right flanks of C57BL/6 mice were injected with1×10⁶E.G7-OVA lymphoma (11 per group). The mice were then challengedthree times with 100 μg OVA s.c., 1.8 μg OVA₂₅₇₋₂₆₄ s.c., and 0.92 nmololigo in AST-008, and sacrificed at 2000 mm³. FIG. 22B is a graph of theresults. *p<0.05 using Two-way ANOVA.

FIGS. 23A-23B show that AST-008 exhibits superior anti-tumor activityand longer survival than CpG oligodeoxynucleotides. The graphs show thetumor volume (FIG. 23A) and percent survival (FIG. 23B) after C57BL/6mice were injected with 1×10⁶ E.G7-OVA lymphoma in their right flanks(11 per group) and then were challenged three times with PBS, PBS andOVA, CpG 1826 and OVA, or AST-008 and OVA. *p<0.05.

DETAILED DESCRIPTION

This invention is not limited in its application to the details ofconstruction and the arrangement of components set forth in thefollowing description or illustrated in the drawings. The invention iscapable of other embodiments and of being practiced or of being carriedout in various ways. Also, the phraseology and terminology used hereinis for the purpose of description and should not be regarded aslimiting. The use of “including,” “comprising,” or “having,”“containing,” “involving,” and variations thereof herein, is meant toencompass the items listed thereafter and equivalents thereof as well asadditional items.

The present invention, in some aspects, overcomes several major hurdlesencountered by conventional TLR 3, TLR 7/8, and TLR 9 agonists byachieving faster activation, creating a multivalent structure, changingcellular distribution, and facilitating simple and scalable synthesis ofvarious adjuvant and antigen-containing structures, among others. Theconstructs of the invention result in more effective vaccines forprophylactic or therapeutic uses in treating a wide variety ofdiseases/infections including, for example, AIDS, malaria, chlamydia,campylobacter, cytomegalovirus, dengue, Epstein-Barr mononucleosis, footand mouth disease, rabies, Helicobacter pylori gastric ulcers, hepatitisA, B, C, herpes simplex, influenza, leishmaniasis, cholera, diphtheria,Haemophilus influenza, meningococcal meningitis, plague, pneumococcalpneumonia, tetanus, typhoid fever, respiratory synctial virus,rhinovirus, schistosomiasis, shigella, streptococcus group A and B,tuberculosis, vibrio cholera, salmonella, aspergillus, blastomyces,histoplasma, candida, cryptococcus, pneumocystis, and urinary tractinfections; various food allergies such as peanut, fruit, garlic, oats,meat, milk, fish, shellfish, soy, tree nut, wheat, gluten, egg,sulphites; various drug allergies such as to tetracycline, Dilantin,carbamazepine, penicillins, cephalosporins, sulfonamides, NSAIDs,intravenous contrast dye, local anesthetics; autoimmune diseases such asmultiple sclerosis, lupus, inflammatory bowel disease, Crohn's disease,ulcerative colitis, asthma, and COPD; and cancers such as melanoma,breast cancer, prostate cancer, bladder cancer, NSCLC, glioblastomamultiforme, among others. A set of exemplary nanoscale constructs of theinvention is shown in the schematic of

FIGS. 1A-1B. The platform described herein is useful for loading one ormultiple agonists of nucleic acid-interacting complexes (A) or one ormultiple agonists of nucleic acid-interacting complexes and antigen (B).Optimization of: (1) nucleic acid interacting complex, such as a TLRthat is targeted (TLR 3, 7/8, and/or 9), (2) density of agonist ofnucleic acid-interacting complexes, (3) antigen density, (4) multipleantigen presentation, (5) core composition, size, and charge, and (6)core linker chemistry “L”, and (7) agonist chemical structure isexpected to yield novel paradigms in vaccine development. In particular,agonists of nucleic acid-interacting complexes include double strandedRNA (such as poly(I:C), TLR 3), single stranded RNA (such as strandscontaining UUG-motifs, TLR 7/8), and unmethylated deoxyribonucleic acidand derivatives (such as strands containing CpG motifs).

Aspects of the invention relate to nanoscale constructs. A nanoscaleconstruct refers to a nanometer sized construct having one or morenucleic acids held in a geometrical position. The nanoscale constructtypically is referred to as a corona of a set of nucleic acids. Acorona, as used herein, refers to an exterior shell composed of nucleicacid molecules. The corona may have a nanoparticle core composed ofnucleic acids or other materials, such as metals. Alternatively, thecorona may simply be a set of nucleic acids arranged in a geometricshape with a hollow core, i.e. a 3-dimensionally shaped layer of nucleicacids. Typically, but not always, the corona has a spherical shape.

In the instance, when the corona includes a nanoparticle core thenucleic acids may be linked directly to the core. Some or all of thenucleic acids may be linked to other nucleic acids either directly orindirectly through a covalent or non-covalent linkage. The linkage ofone nucleic acid to another nucleic acid may be in addition to oralternatively to the linkage of that nucleic acid to a core. One or moreof the nucleic acids may also be linked to other molecules such as anantigen.

When the corona does not include a nanoparticle core, the nucleic acidsmay be linked to one another either directly or indirectly through acovalent or non-covalent linkage. In some embodiments the corona thatdoes not include a nanoparticle core may be formed by layering thenucleic acids on a lattice or other dissolvable structure and thendissolving the lattice or other structure to produce an empty center.

As used herein, the nanoscale construct is a construct having an averagediameter on the order of nanometers (i.e., between about 1 nm and about1 micrometer. For example, in some instances, the diameter of thenanoparticle is from about 1 nm to about 250 nm in mean diameter, about1 nm to about 240 nm in mean diameter, about 1 nm to about 230 nm inmean diameter, about 1 nm to about 220 nm in mean diameter, about 1 nmto about 210 nm in mean diameter, about 1 nm to about 200 nm in meandiameter, about 1 nm to about 190 nm in mean diameter, about 1 nm toabout 180 nm in mean diameter, about 1 nm to about 170 ran in meandiameter, about 1 nm to about 160 nm in mean diameter, about 1 nm toabout 150 nm in mean diameter, about 1 nm to about 140 nm in meandiameter, about 1 nm to about 130 nm in mean diameter, about 1 nm toabout 120 nm in mean diameter, about 1 nm to about 110 nm in meandiameter, about 1 nm to about 100 nm in mean diameter, about 1 nm toabout 90 nm in mean diameter, about 1 nm to about 80 nm in meandiameter, about 1 nm to about 70 nm in mean diameter, about 1 nm toabout 60 nm in mean diameter, about 1 nm to about 50 nm in meandiameter, about 1 nm to about 40 nm in mean diameter, about 1 nm toabout 30 nm in mean diameter, about 1 nm to about 20 nm in meandiameter, about 1 nm to about 10 nm in mean diameter, about 5 nm toabout 150 nm in mean diameter, about 5 to about 50 nm in mean diameter,about 10 to about 30 nm in mean diameter, about 10 to 150 nm in meandiameter, about 10 to about 100 nm in mean diameter, about 10 to about50 nm in mean diameter, about 30 to about 100 nm in mean diameter, orabout 40 to about 80 nm in mean diameter.

In some instances the corona includes a nanoparticle core that isattached to one or more agonists of nucleic acid-interacting complexesand/or antigens. As used herein, a nanoparticle core refers to thenanoparticle component of a nanoparticle construct, without any attachedmodalities. In some instances, the nanoparticle core is metallic. Itshould be appreciated that the nanoparticle core can comprise any metal.Several non-limiting examples of metals include gold, silver, platinum,aluminum, palladium, copper, cobalt, indium, nickel and mixturesthereof. In some embodiments, the nanoparticle core comprises gold. Forexample, the nanoparticle core can be a lattice structure includingdegradable gold. Nanoparticles can also comprise semiconductor andmagnetic materials.

Non-limiting examples of nanoparticles compatible with aspects of theinvention are described in and incorporated by reference from: US PatentNo. 7,238,472, US Patent Publication No. 2003/0147966, US PatentPublication No. 2008/0306016, US Patent Publication No. 2009/0209629, USPatent Publication No. 2010/0136682, US Patent Publication No.2010/0184844, US Patent Publication No. 2010/0294952, US PatentPublication No. 2010/0129808, US Patent Publication No. 2010/0233270, USPatent Publication No. 2011/0111974, PCT Publication No. WO 2002/096262,PCT Publication No. WO 2003/08539, PCT Publication No. WO 2006/138145,PCT Publication No. WO 2008/127789, PCT Publication No. WO 2008/098248,PCT Publication No. WO 2011/079290, PCT Publication No. WO 2011/053940,PCT Publication No. WO 2011/017690 and PCT Publication No. WO2011/017456. Nanoparticles associated with the invention can besynthesized according to any means known in the art or can be obtainedcommercially. For example, several non-limiting examples of commercialsuppliers of nanoparticles include: Ted Pella, Inc., Redding, Calif.,Nanoprobes, Inc., Yaphank, N.Y., Vacuum Metallurgical Co,. Ltd., Chiba,Japan and Vector Laboratories, Inc., Burlington, Calif.

Agonists of Nucleic Acid-Interacting Complexes

A nucleic acid-interacting complex as used herein refers to a moleculeor complex of molecules that interact with a nucleic acid molecule andare stimulated to produce an immune response in response to thatinteraction. The molecule or complex of molecules may be a receptor, forinstance. In some embodiments a nucleic acid-interacting complex is apattern recognition receptor (PRR) complex. PRRs are a primitive part ofthe immune system composed of proteins expressed by cells of the innateimmune system to identify pathogen-associated molecular patterns(PAMPs), which are associated with microbial pathogens or cellularstress, as well as damage-associated molecular patterns (DAMPs), whichare associated with cell components released during cell damage. PRRsinclude but are not limited to membrane-bound PRRs, such as receptorkinases, toll-like receptors (TLR), and C-type lectin Receptors (CLR)(mannose receptors and asialoglycoprotein receptors); Cytoplasmic PRRssuch as RIG-I-like receptors (RLR), RNA Helicases, Plant PRRs, and NonRDkinases; and secreted PRRs.

Nucleic acid-interacting complexes include but are not limited to TLRs,RIG-I, transcription factors, cellular translation machinery, cellulartranscription machinery, nucleic-acid acting enzymes, and nucleic acidassociating autoantigens. Nucleic acid molecules that are agonists of anucleic acid-interacting complex include but are not limited to TLRagonists, and agonists of RIG-I, transcription factors, cellulartranslation machinery, cellular transcription machinery, nucleic-acidacting enzymes, and nucleic acid associating autoantigens.

In some embodiments an agonist of a nucleic acid-interacting complex isa TLR agonist. A TLR agonist, as used herein is a nucleic acid moleculethat interacts with and stimulates the activity of a TLR.

Toll-like receptors (TLRs) are a family of highly conserved polypeptidesthat play a critical role in innate immunity in mammals. At least tenfamily members, designated TLR1-TLR10, have been identified. Thecytoplasmic domains of the various TLRs are characterized by aToll-interleukin 1 (IL-1) receptor (TIR) domain. Medzhitov R et al.(1998) Mol Cell 2:253-8. Recognition of microbial invasion by TLRstriggers activation of a signaling cascade that is evolutionarilyconserved in Drosophila and mammals. The TIR domain-containing adaptorprotein MyD88 has been reported to associate with TLRs and to recruitIL-1 receptor-associated kinase (IRAK) and tumor necrosis factor (TNF)receptor-associated factor 6 (TRAF6) to the TLRs. The MyD88-dependentsignaling pathway is believed to lead to activation of NF-κBtranscription factors and c-Jun NH2 terminal kinase (Jnk)mitogen-activated protein kinases (MAPKs), critical steps in immuneactivation and production of inflammatory cytokines. For a review, seeAderem A et al. (2000) Nature 406:782-87.

TLRs are believed to be differentially expressed in various tissues andon various types of immune cells. For example, human TLR7 has beenreported to be expressed in placenta, lung, spleen, lymph nodes, tonsiland on plasmacytoid precursor dendritic cells (pDCs). Chuang T-H et al.(2000) Eur Cytokine Netw 11:372-8); Kadowaki Net al. (2001) J Exp Med194:863-9. Human TLR8 has been reported to be expressed in lung,peripheral blood leukocytes (PBL), placenta, spleen, lymph nodes, and onmonocytes. Kadowaki N et al. (2001) J Exp Med 194:863-9; Chuang T-H etal. (2000) Eur Cytokine Netw 11:372-8. Human TLR9 is reportedlyexpressed in spleen, lymph nodes, bone marrow, PBL, and on pDCs, and Bcells. Kadowaki N et al. (2001) J Exp Med 194:863-9; Bauer S et al.(2001) Proc Natl Acad Sci USA 98:9237-42; Chuang T-H et al. (2000) EurCytokine Netw 11:372-8.

Nucleotide and amino acid sequences of human and murine TLR7 are known.See, for example, GenBank Accession Nos. AF240467, AF245702, NM_016562,AF334942, NM_133211; and AAF60188, AAF78035, NP_057646, AAL73191, andAAL73192, the contents of all of which are incorporated herein byreference. Human TLR7 is reported to be 1049 amino acids long. MurineTLR7 is reported to be 1050 amino acids long. TLR7 polypeptides includean extracellular domain having a leucine-rich repeat region, atransmembrane domain, and an intracellular domain that includes a TIRdomain.

Nucleotide and amino acid sequences of human and murine TLR8 are known.See, for example, GenBank Accession Nos. AF246971, AF245703, NM_016610,XM_045706, AY035890, NM_133212; and AAF64061, AAF78036, NP_057694,XP_045706, AAK62677, and NP_573475, the contents of all of which isincorporated herein by reference. Human TLR8 is reported to exist in atleast two isoforms, one 1041 amino acids long and the other 1059 aminoacids long. Murine TLR8 is 1032 amino acids long. TLR8 polypeptidesinclude an extracellular domain having a leucine-rich repeat region, atransmembrane domain, and an intracellular domain that includes a TIRdomain.

Nucleotide and amino acid sequences of human and murine TLR9 are known.See, for example, GenBank Accession Nos. NM_017442, AF259262, AB045180,AF245704, AB045181, AF348140, AF314224, NM_031178; and NP_059138,AAF72189, BAB19259, AAF78037, BAB19260, AAK29625, AAK28488, andNP_112455, the contents of all of which are incorporated herein byreference. Human TLR9 is reported to exist in at least two isoforms, one1032 amino acids long and the other 1055 amino acids. Murine TLR9 is1032 amino acids long. TLR9 polypeptides include an extracellular domainhaving a leucine-rich repeat region, a transmembrane domain, and anintracellular domain that includes a TIR domain.

As used herein, the term “TLR signaling” refers to any aspect ofintracellular signaling associated with signaling through a TLR. As usedherein, the term “TLR-mediated immune response” refers to the immuneresponse that is associated with TLR signaling.

A TLR7-mediated immune response is a response associated with TLR7signaling. TLR7-mediated immune response is generally characterized bythe induction of IFN-α and IFN-inducible cytokines such as IP-10 andI-TAC. The levels of cytokines IL-1 α/β, IL-6, IL-8, MIP-1 α/β and MIP-3α/β induced in a TLR7-mediated immune response are less than thoseinduced in a TLR8-mediated immune response.

A TLR8-mediated immune response is a response associated with TLR8signaling. This response is further characterized by the induction ofpro-inflammatory cytokines such as IFN-γ, IL-12p40/70, TNF-α, IL-1 α/β,IL-6, IL-8, MIP-1 α/β and MIP-3 α/β.

A TLR9-mediated immune response is a response associated with TLR9signaling. This response is further characterized at least by theproduction/secretion of IFN-γ and IL-12, albeit at levels lower than areachieved via a TLR8-mediated immune response.

As used herein, a “TLR7/8 agonist” collectively refers to any nucleicacid that is capable of increasing TLR7 and/or TLR8 signaling (i.e., anagonist of TLR7 and/or TLR8). Some TLR7/8 ligands induce TLR7 signalingalone (e.g., TLR7 specific agonists), some induce TLR8 signaling alone(e.g., TLR8 specific agonists), and others induce both TLR7 and TLR8signaling.

The level of TLR7 or TLR8 signaling may be enhanced over a pre-existinglevel of signaling or it may be induced over a background level ofsignaling. TLR7 ligands include, without limitation, guanosine analoguessuch as C8-substituted guanosines, mixtures of ribonucleosidesconsisting essentially of G and U, guanosine ribonucleotides and RNA orRNA-like molecules (PCT/US03/10406), and adenosine-based compounds(e.g., 6-amino-9-benzyl-2-(3-hydroxy-propoxy)-9H-purin-8-ol, and similarcompounds made by Sumitomo (e.g., CL-029)).

As used herein, the term “guanosine analogues” refers to aguanosine-like nucleotide (excluding guanosine) having a chemicalmodification involving the guanine base, guanosine nucleoside sugar, orboth the guanine base and the guanosine nucleoside sugar. Guanosineanalogues specifically include, without limitation, 7-deaza-guanosine.

Guanosine analogues further include C8-substituted guanosines such as7-thia-8-oxoguanosine (immunosine), 8-mercaptoguanosine,8-bromoguanosine, 8-methylguanosine, 8-oxo-7,8-dihydroguanosine,C8-arylamino-2′-deoxyguanosine, C8-propynyl-guanosine, C8- andN7-substituted guanine ribonucleosides such as 7-allyl-8-oxoguanosine(loxoribine) and 7-methyl-8-oxoguanosine, 8-aminoguanosine,8-hydroxy-2′-deoxyguanosine, 8-hydroxyguanosine, and 7-deaza8-substituted guanosine.

TLR8 ligands include mixtures of ribonucleosides consisting essentiallyof G and U, guanosine ribonucleotides and RNA or RNA-like molecules(PCT/US03/10406). Additional TLR8 ligands are also disclosed in Gordenet al. J. Immunol. 2005, 174:1259-1268). As used herein, the term “TLR9agonist” refers to any agent that is capable of increasing

TLR9 signaling (i.e., an agonist of TLR9). TLR9 agonists specificallyinclude, without limitation, immunostimulatory nucleic acids, and inparticular CpG immunostimulatory nucleic acids.

As used herein, the term “immunostimulatory CpG nucleic acids” or“immunostimulatory CpG oligonucleotides” refers to any CpG-containingnucleic acid that is capable of activating an immune cell. At least theC of the CpG dinucleotide is typically, but not necessarily,unmethylated. Immunostimulatory CpG nucleic acids are described in anumber of issued patents and published patent applications, includingU.S. Pat. Nos. 6,194,388; 6,207,646; 6,218,371; 6,239,116; 6,339,068;6,406,705; and 6,429,199.

In some embodiments the agonists of nucleic acid-interacting complexesis an immunostimulatory oligonucleotide. An “immunostimulatoryoligonucleotide” as used herein is any nucleic acid (DNA or RNA)containing an immunostimulatory motif or backbone that is capable ofinducing an immune response. An induction of an immune response refersto any increase in number or activity of an immune cell, or an increasein expression or absolute levels of an immune factor, such as acytokine. Immune cells include, but are not limited to, NK cells, CD4+Tlymphocytes, CD8+T lymphocytes, B cells, dendritic cells, macrophage andother antigen-presenting cells. Cytokines include, but are not limitedto, interleukins, TNF-α, IFN-α,β and γ, Flt-ligand, and co-stimulatorymolecules. Immunostimulatory motifs include, but are not limited to CpGmotifs and T-rich motifs.

A non-limiting set of immunostimulatory oligonucleotides includes:

dsRNA (TLR 3): poly(A:C) and poly(I:C) ssRNA (TLR7/8): (SEQ ID NO: 13)CCGUCUGUUGUGUGACUC (SEQ ID NO: 14) GCCACCGAGCCGAAGGCACC (SEQ ID NO: 15)UAUAUAUAUAUAUAUAUAUA (SEQ ID NO: 16) UUAUUAUUAUUAUUAUUAUU(SEQ ID NO: 17) UUUUAUUUUAUUUUAUUUUA (SEQ ID NO: 18)UGUGUGUGUGUGUGUGUGUG (SEQ ID NO: 19) UUGUUGUUGUUGUUGUUGUU(SEQ ID NO: 20) UUUGUUUGUUUGUUUGUUUG (SEQ ID NO: 21)UUAUUUAUUUAUUUAUUUAUUUAU (SEQ ID NO: 22) UUGUUUGUUUGUUUGUUUGUUUGU(SEQ ID NO: 23) GCCCGUCUGUUGUGUGACUC (SEQ ID NO: 24) GUCCUUCAAGUCCUUCAADNA (TLR9): (SEQ ID NO: 25) GGTGCATCGATGCAGGGGGG (SEQ ID NO: 26)TCCATGGACGTTCCTGAGCGTT (SEQ ID NO: 27) TCGTCGTTCGAACGACGTTGAT(SEQ ID NO: 28) TCGTCGACGATCCGCGCGCGCG (SEQ ID NO: 29)GGGGTCAACGTTGAGGGGGG (SEQ ID NO: 30) TCGTCGTTTTGTCGTTTTGTCGTT(SEQ ID NO: 31) TCGTCGTTGTCGTTTTGTCGTT (SEQ ID NO: 32)GGGGGACGATCGTCGGGGGG (SEQ ID NO: 33) GGGGACGACGTCGTGGGGGGG(SEQ ID NO: 34) TCGTCGTTTTCGGCGCGCGCCG (SEQ ID NO: 35)TCGTCGTCGTTCGAACGACGTTGAT

The immunostimulatory oligonucleotides may be linked to the core or toone another or to other molecules such an antigens. For instance, theoligonucleotides may be conjugated to a linker via the 5′ end or the 3′end. E.g. [Sequence, 5′-3′]-Linker or Linker-[Sequence, 5′-3′].

The terms “oligonucleotide” and “nucleic acid” are used interchangeablyto mean multiple nucleotides (i.e., molecules comprising a sugar (e.g.,ribose or deoxyribose) linked to a phosphate group and to anexchangeable organic base, which is either a substituted pyrimidine(e.g., cytosine (C), thymidine (T) or uracil (U)) or a substitutedpurine (e.g., adenine (A) or guanine (G)). Thus, the term embraces bothDNA and RNA oligonucleotides. The terms shall also includepolynucleosides (i.e., a polynucleotide minus the phosphate) and anyother organic base containing polymer. Oligonucleotides can be obtainedfrom existing nucleic acid sources (e.g., genomic or cDNA), but arepreferably synthetic (e.g., produced by nucleic acid synthesis). Apolynucleotide of the nanoscale construct and optionally attached to ananoparticle core can be single stranded or double stranded. A doublestranded polynucleotide is also referred to herein as a duplex.Double-stranded oligonucleotides of the invention can comprise twoseparate complementary nucleic acid strands.

As used herein, “duplex” includes a double-stranded nucleic acidmolecule(s) in which complementary sequences are hydrogen bonded to eachother. The complementary sequences can include a sense strand and anantisense strand. The antisense nucleotide sequence can be identical orsufficiently identical to the target gene to mediate effective targetgene inhibition (e.g., at least about 98% identical, 96% identical, 94%,90% identical, 85% identical, or 80% identical) to the target genesequence.

A double-stranded polynucleotide can be double-stranded over its entirelength, meaning it has no overhanging single-stranded sequences and isthus blunt-ended. In other embodiments, the two strands of thedouble-stranded polynucleotide can have different lengths producing oneor more single-stranded overhangs. A double-stranded polynucleotide ofthe invention can contain mismatches and/or loops or bulges. In someembodiments, it is double-stranded over at least about 70%, 80%, 90%,95%, 96%, 97%, 98% or 99% of the length of the oligonucleotide. In someembodiments, the double-stranded polynucleotide of the inventioncontains at least or up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, or 15 mismatches.

Polynucleotides associated with the invention can be modified such as atthe sugar moiety, the phosphodiester linkage, and/or the base. As usedherein, “sugar moieties” includes natural, unmodified sugars, includingpentose, ribose and deoxyribose, modified sugars and sugar analogs.Modifications of sugar moieties can include replacement of a hydroxylgroup with a halogen, a heteroatom, or an aliphatic group, and caninclude functionalization of the hydroxyl group as, for example, anether, amine or thiol.

Modification of sugar moieties can include 2′-O-methyl nucleotides,which are referred to as “methylated.” In some instances,polynucleotides associated with the invention may only contain modifiedor unmodified sugar moieties, while in other instances, polynucleotidescontain some sugar moieties that are modified and some that are not.

In some instances, modified nucleomonomers include sugar- orbackbone-modified ribonucleotides. Modified ribonucleotides can containa non-naturally occurring base such as uridines or cytidines modified atthe 5′-position, e.g., 5′-(2-amino)propyl uridine and 5′-bromo uridine;adenosines and guanosines modified at the 8-position, e.g., 8-bromoguanosine; deaza nucleotides, e.g., 7-deaza-adenosine; and N-alkylatednucleotides, e.g., N6-methyl adenosine. Also, sugar-modifiedribonucleotides can have the 2′-OH group replaced by an H, alkoxy (orOR), R or alkyl, halogen, SH, SR, amino (such as NH₂, NHR, NR_(2,)), orCN group, wherein R is lower alkyl, alkenyl, or alkynyl. In someembodiments, modified ribonucleotides can have the phosphodiester groupconnecting to adjacent ribonucleotides replaced by a modified group,such as a phosphorothioate group.

In some aspects, 2′-O-methyl modifications can be beneficial forreducing undesirable cellular stress responses, such as the interferonresponse to double-stranded nucleic acids. Modified sugars can includeD-ribose, 2′-O-alkyl (including 2′-O-methyl and 2′-O-ethyl), i.e.,2′-alkoxy, 2′-amino, 2′-S-alkyl, 2′-halo (including 2′-fluoro),2′-methoxyethoxy, 2′-allyloxy (—OCH₂CH═CH₂), 2′-propargyl, 2′-propyl,ethynyl, ethenyl, propenyl, and cyano and the like. The sugar moiety canalso be a hexose.

The term “alkyl” includes saturated aliphatic groups, includingstraight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl,hexyl, heptyl, octyl, nonyl, decyl, etc.), branched-chain alkyl groups(isopropyl, tert-butyl, isobutyl, etc.), cycloalkyl (alicyclic) groups(cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), alkylsubstituted cycloalkyl groups, and cycloalkyl substituted alkyl groups.In some embodiments, a straight chain or branched chain alkyl has 6 orfewer carbon atoms in its backbone (e.g., C₁-C₆ for straight chain,C₃-C₆ for branched chain), and more preferably 4 or fewer. Likewise,preferred cycloalkyls have from 3-8 carbon atoms in their ringstructure, and more preferably have 5 or 6 carbons in the ringstructure. The term C₁-C₆ includes alkyl groups containing 1 to 6 carbonatoms.

Unless otherwise specified, the term alkyl includes both “unsubstitutedalkyls” and “substituted alkyls,” the latter of which refers to alkylmoieties having independently selected substituents replacing a hydrogenon one or more carbons of the hydrocarbon backbone. Such substituentscan include, for example, alkenyl, alkynyl, halogen, hydroxyl,alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano,amino (including alkyl amino, dialkylamino, arylamino, diarylamino, andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.Cycloalkyls can be further substituted, e.g., with the substituentsdescribed above. An “alkylaryl” or an “arylalkyl” moiety is an alkylsubstituted with an aryl (e.g., phenylmethyl (benzyl)). The term “alkyl”also includes the side chains of natural and unnatural amino acids. Theterm “n-alkyl” means a straight chain (i.e., unbranched) unsubstitutedalkyl group.

The term “alkenyl” includes unsaturated aliphatic groups analogous inlength and possible substitution to the alkyls described above, but thatcontain at least one double bond. For example, the term “alkenyl”includes straight-chain alkenyl groups (e.g., ethylenyl, propenyl,butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, etc.),branched-chain alkenyl groups, cycloalkenyl (alicyclic) groups(cyclopropenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl,cyclooctenyl), alkyl or alkenyl substituted cycloalkenyl groups, andcycloalkyl or cycloalkenyl substituted alkenyl groups. In someembodiments, a straight chain or branched chain alkenyl group has 6 orfewer carbon atoms in its backbone (e.g., C₂-C₆ for straight chain,C₃-C₆ for branched chain). Likewise, cycloalkenyl groups may have from3-8 carbon atoms in their ring structure, and more preferably have 5 or6 carbons in the ring structure. The term C2-C6 includes alkenyl groupscontaining 2 to 6 carbon atoms.

Unless otherwise specified, the term alkenyl includes both“unsubstituted alkenyls” and “substituted alkenyls,” the latter of whichrefers to alkenyl moieties having independently selected substituentsreplacing a hydrogen on one or more carbons of the hydrocarbon backbone.Such substituents can include, for example, alkyl groups, alkynylgroups, halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety.

The term “hydrophobic modifications’ refers to modification of basessuch that overall hydrophobicity is increased and the base is stillcapable of forming close to regular Watson-Crick interactions.Non-limiting examples of base modifications include 5-position uridineand cytidine modifications like phenyl, 4-pyridyl, 2-pyridyl, indolyl,and isobutyl, phenyl (C₆H₅OH); tryptophanyl (C₈H₆N)CH₂CH(NH₂)CO),Isobutyl, butyl, aminobenzyl; phenyl; naphthyl,

The term “heteroatom” includes atoms of any element other than carbon orhydrogen. In some embodiments, preferred heteroatoms are nitrogen,oxygen, sulfur and phosphorus. The term “hydroxy” or “hydroxyl” includesgroups with an—OH or —O^(—) (with an appropriate counterion). The term“halogen” includes fluorine, bromine, chlorine, iodine, etc. The term“perhalogenated” generally refers to a moiety wherein all hydrogens arereplaced by halogen atoms.

The term “substituted” includes independently selected substituentswhich can be placed on the moiety and which allow the molecule toperform its intended function. Examples of substituents include alkyl,alkenyl, alkynyl, aryl, (CR′R″)₀₋₃NR′R″, (CR′R″)₀₋₃CN, NO2, halogen,(CR′R″)₀₋₃C(halogen)₃, (CR′R″)₀₋₃CH(halogen)2, (CR′R″)₀₋₃CH2(halogen),(CR′R″)₀₋₃CONR′R″, (CR′R″)₀₋₃S(O)₁₋₂NR′R″, (CR′R″)₀₋₃CHO,(CR′R″)₀₋₃O(CR′R″)₀₋₃H, (CR′R″)₀₋₃S(O)₀₋₂R′, (CR′R″)₀₋₃O(CR′R″)₀₋₃H,(CR′R″)₀₋₃COR′, (CR′R″)₀₋₃CO₂R′, or (CR′R″)₀₋₃′ groups; wherein each R′and R″ are each independently hydrogen, a C₁-C₅ alkyl, C₂-C₅ alkenyl,C₂-C₅ alkynyl, or aryl group, or R′ and R″ taken together are abenzylidene group or a —(CH₂)₂O(CH2)₂— group.

The term “amine” or “amino” includes compounds or moieties in which anitrogen atom is covalently bonded to at least one carbon or heteroatom.The term “alkyl amino” includes groups and compounds wherein thenitrogen is bound to at least one additional alkyl group. The term“dialkyl amino” includes groups wherein the nitrogen atom is bound to atleast two additional alkyl groups.

The term “ether” includes compounds or moieties which contain an oxygenbonded to two different carbon atoms or heteroatoms. For example, theterm includes “alkoxyalkyl,” which refers to an alkyl, alkenyl, oralkynyl group covalently bonded to an oxygen atom which is covalentlybonded to another alkyl group.

The term “base” includes the known purine and pyrimidine heterocyclicbases, deazapurines, and analogs (including heterocyclic substitutedanalogs, e.g., aminoethyoxy phenoxazine), derivatives (e.g., 1-alkyl-,1-alkenyl-, heteroaromatic- and 1-alkynyl derivatives) and tautomersthereof. Examples of purines include adenine, guanine, inosine,diaminopurine, and xanthine and analogs (e.g., 8-oxo-N⁶-methyladenine or7-diazaxanthine) and derivatives thereof. Pyrimidines include, forexample, thymine, uracil, and cytosine, and their analogs (e.g.,5-methylcytosine, 5-methyluracil, 5-(1-propynyl)uracil,5-(1-propynyl)cytosine and 4,4-ethanocytosine). ethanocytosine). Otherexamples of suitable bases include non-purinyl and non-pyrimidinyl basessuch as 2-aminopyridine and triazines.

In some aspects, the nucleomonomers of a polynucleotide of the inventionare RNA nucleotides, including modified RNA nucleotides. The term“nucleoside” includes bases which are covalently attached to a sugarmoiety, preferably ribose or deoxyribose. Examples of preferrednucleosides include ribonucleosides and deoxyribonucleosides.Nucleosides also include bases linked to amino acids or amino acidanalogs which may comprise free carboxyl groups, free amino groups, orprotecting groups. Suitable protecting groups are well known in the art(see P. G. M. Wuts and T. W. Greene, “Protective Groups in OrganicSynthesis”, 2^(nd) Ed., Wiley-Interscience, New York, 1999).

The term “nucleotide” includes nucleosides which further comprise aphosphate group or a phosphate analog.

As used herein, the term “linkage” includes a naturally occurring,unmodified phosphodiester moiety (—O—(PO^(2—))—O—) that covalentlycouples adjacent nucleomonomers. As used herein, the term “substitutelinkage” includes any analog or derivative of the native phosphodiestergroup that covalently couples adjacent nucleomonomers. Substitutelinkages include phosphodiester analogs, e.g., phosphorothioate,phosphorodithioate, and P-ethyoxyphosphodiester, P-ethoxyphosphodiester,P-alkyloxyphosphotriester, methylphosphonate, and nonphosphoruscontaining linkages, e.g., acetals and amides. Such substitute linkagesare known in the art (e.g., Bjergarde et al. 1991. Nucleic Acids Res.19:5843; Caruthers et al. 1991. Nucleosides Nucleotides. 10:47). Incertain embodiments, non-hydrolizable linkages are preferred, such asphosphorothioate linkages.

In some aspects, polynucleotides of the invention comprise 3′ and 5′termini (except for circular oligonucleotides). The 3′ and 5′ termini ofa polynucleotide can be substantially protected from nucleases, forexample, by modifying the 3′ or 5′ linkages (e.g., U.S. Pat. No.5,849,902 and WO 98/13526). Oligonucleotides can be made resistant bythe inclusion of a “blocking group.” The term “blocking group” as usedherein refers to substituents (e.g., other than OH groups) that can beattached to oligonucleotides or nucleomonomers, either as protectinggroups or coupling groups for synthesis (e.g., FITC, propyl(CH₂—CH₂—CH₃), glycol (—O—CH₂—CH₂—O—) phosphate (PO₃ ^(2—)), hydrogenphosphonate, or phosphoramidite). “Blocking groups” also include “endblocking groups” or “exonuclease blocking groups” which protect the 5′and 3′ termini of the oligonucleotide, including modified nucleotidesand non-nucleotide exonuclease resistant structures.

Exemplary end-blocking groups include cap structures (e.g., a7-methylguanosine cap), inverted nucleomonomers, e.g., with 3′-3′ or5′-5′ end inversions (see, e.g., Ortiagao et al. 1992. Antisense Res.Dev. 2:129), methylphosphonate, phosphoramidite, non-nucleotide groups(e.g., non-nucleotide linkers, amino linkers, conjugates) and the like.The 3′ terminal nucleomonomer can comprise a modified sugar moiety. The3′ terminal nucleomonomer comprises a 3′-0 that can optionally besubstituted by a blocking group that prevents 3′-exonuclease degradationof the oligonucleotide. For example, the 3′-hydroxyl can be esterifiedto a nucleotide through a 3′→3′ internucleotide linkage. For example,the alkyloxy radical can be methoxy, ethoxy, or isopropoxy, andpreferably, ethoxy. Optionally, the 3′→31′linked nucleotide at the 3′terminus can be linked by a substitute linkage. To reduce nucleasedegradation, the 5′ most 3′→5′ linkage can be a modified linkage, e.g.,a phosphorothioate or a P-alkyloxyphosphotriester linkage. Preferably,the two 5′ most 3′→5′ linkages are modified linkages. Optionally, the 5′terminal hydroxy moiety can be esterified with a phosphorus containingmoiety, e.g., phosphate, phosphorothioate, or P-ethoxyphosphate.

In some aspects, polynucleotides can comprise both DNA and RNA.

In some aspects, at least a portion of the contiguous polynucleotidesare linked by a substitute linkage, e.g., a phosphorothioate linkage.The presence of substitute linkages can improve pharmacokinetics due totheir higher affinity for serum proteins.

CpG sequences, while relatively rare in human DNA are commonly found inthe DNA of infectious organisms such as bacteria. The human immunesystem has apparently evolved to recognize CpG sequences as an earlywarning sign of infection and to initiate an immediate and powerfulimmune response against invading pathogens without causing adversereactions frequently seen with other immune stimulatory agents. Thus CpGcontaining nucleic acids, relying on this innate immune defensemechanism can utilize a unique and natural pathway for immune therapy.The effects of CpG nucleic acids on immune modulation have beendescribed extensively in U.S. Pat. No. 6,194,388, and published patentapplications, such as PCT US95/01570), PCT/US97/19791, PCT/US98/03678;PCT/US98/10408; PCT/US98/04703; PCT/US99/07335; and PCT/US99/09863.

A “CpG oligonucleotide” is a nucleic acid which includes at least oneunmethylated CpG dinucleotide. In some embodiments, the nucleic acidincludes three or more unmethylated CpG dinucleotides. A nucleic acidcontaining at least one “unmethylated CpG dinucleotide” is a nucleicacid molecule which contains an unmethylated cytosine in acytosine-guanine dinucleotide sequence (i.e. “CpG DNA” or DNA containinga 5′ cytosine followed by 3′ guanosine and linked by a phosphate bond)and activates the immune system.

The immunostimulatory oligonucleotides of the nanoscale construct arepreferably in the range of 6 to 100 bases in length. However, nucleicacids of any size greater than 6 nucleotides (even many kb long) arecapable of inducing an immune response according to the invention ifsufficient immunostimulatory motifs are present. Preferably theimmunostimulatory nucleic acid is in the range of between 8 and 100 andin some embodiments between 8 and 50 or 8 and 30 nucleotides in size.

In some embodiments the immunostimulatory oligonucleotides have amodified backbone such as a phosphorothioate (PS) backbone. In otherembodiments the immunostimulatory oligonucleotides have a phosphodiester(PO) backbone. In yet other embodiments immunostimulatoryoligonucleotides have a mixed PO and PS backbone.

Attachment of Modalities to Nanoparticle Cores

Modalities associated with the invention, including agonists of nucleicacid-interacting complexes and antigens, can be attached to nanoparticlecores by any means known in the art. Methods for attachingoligonucleotides to nanoparticles are described in detail in andincorporated by reference from US Patent Publication No. 2010/0129808.

A nanoparticle can be functionalized in order to attach apolynucleotide. Alternatively or additionally, the polynucleotide can befunctionalized. One mechanism for functionalization is the alkanethiolmethod, whereby oligonucleotides are functionalized with alkanethiols attheir 3′ or 5′ termini prior to attachment to gold nanoparticles ornanoparticles comprising other metals, semiconductors or magneticmaterials. Such methods are described, for example Whitesides,Proceedings of the Robert A. Welch Foundation 39th Conference OnChemical Research Nanophase Chemistry, Houston, Tex., pages 109-121(1995), and Mucic et al. Chem. Commun. 555-557 (1996). Oligonucleotidescan also be attached to nanoparticles using other functional groups suchas phosophorothioate groups, as described in and incorporated byreference from U.S. Pat. No. 5,472,881, or substituted alkylsiloxanes,as described in and incorporated by reference from Burwell, ChemicalTechnology, 4, 370-377 (1974) and Matteucci and Caruthers, J. Am. Chem.Soc., 103, 3185-3191 (1981). In some instances, polynucleotides areattached to nanoparticles by terminating the polynucleotide with a 5′ or3′ thionucleoside. In other instances, an aging process is used toattach polynucleotides to nanoparticles as described in and incorporatedby reference from U.S. Pat. Nos. 6,361,944, 6,506, 569, 6,767,702 and6,750,016 and PCT Publication Nos. WO 1998/004740, WO 2001/000876, WO2001/051665 and WO 2001/073123.

In some instances, the nucleic acid and/or antigen are covalentlyattached to the nanoparticle core, such as through a gold-thiol linkage.A spacer sequence can be included between the attachment site and theuptake control moiety and/or the binding moiety. In some embodiments, aspacer sequence comprises or consists of an oligonucleotide, a peptide,a polymer or an oligoethylene.

Nanoscale constructs can be designed with multiple chemistries. Forexample, a DTPA (dithiol phosphoramidite) linkage can be used. The DTPAresists intracellular release of flares by thiols and can serve toincrease signal to noise ratio.

The conjugates produced by the methods described herein are considerablymore stable than those produced by other methods. This increasedstability is due to the increased density of the oligonucleotides on thesurfaces of a nanoparticle core or forming the surface of the corona. Byperforming the salt additions in the presence of a surfactant, forexample approximately 0.01% sodium dodecylsulfate (SDS), Tween, orpolyethylene glycol (PEG), the salt aging process can be performed inabout an hour.

The surface density may depend on the size and type of nanoparticles andon the length, sequence and concentration of the oligonucleotides. Asurface density adequate to make the nanoparticles stable and theconditions necessary to obtain it for a desired combination ofnanoparticles and oligonucleotides can be determined empirically.Generally, a surface density of at least 10 picomoles/cm will beadequate to provide stable nanoparticle-oligonucleotide conjugates.Preferably, the surface density is at least 15 picomoles/cm. Since theability of the oligonucleotides of the conjugates to hybridize withtargets may be diminished if the surface density is too great, thesurface density optionally is no greater than about 35-40 picomoles/cm².Methods are also provided wherein the oligonucleotide is bound to thenanoparticle at a surface density of at least 10 pmol/cm², at least 15pmol/cm², at least 20 pmol/cm², at least 25 pmol/cm², at least 30pmol/cm², at least 35 pmol/cm², at least 40 pmol/cm², at least 45pmol/cm, at least 50 pmol/cm², or 50 pmol/cm² or more.

Therapeutics

Aspects of the invention relate to delivery of nanoscale constructs to asubject for therapeutic and/or diagnostic use. The particles may beadministered alone or in any appropriate pharmaceutical carrier, such asa liquid, for example saline, or a powder, for administration in vivo.They can also be co-delivered with larger carrier particles or withinadministration devices. The particles may be formulated. Theformulations of the invention can be administered in pharmaceuticallyacceptable solutions, which may routinely contain pharmaceuticallyacceptable concentrations of salt, buffering agents, preservatives,compatible carriers, adjuvants, and optionally other therapeuticingredients. In some embodiments, nanoscale constructs associated withthe invention are mixed with a substance such as a lotion (for example,aquaphor) and are administered to the skin of a subject, whereby thenanoscale constructs are delivered through the skin of the subject. Itshould be appreciated that any method of delivery of nanoparticles knownin the art may be compatible with aspects of the invention.

For use in therapy, an effective amount of the particles can beadministered to a subject by any mode that delivers the particles to thedesired cell. Administering pharmaceutical compositions may beaccomplished by any means known to the skilled artisan. Routes ofadministration include but are not limited to oral, parenteral,intramuscular, intravenous, subcutaneous, mucosal, intranasal,sublingual, intratracheal, inhalation, ocular, vaginal, dermal, rectal,and by direct injection.

Thus, the invention in one aspect involves the finding that agonists ofnucleic acid-interacting complexes are highly effective in mediatingimmune stimulatory effects. These agonists of nucleic acid-interactingcomplexes are useful therapeutically and prophylactically forstimulating the immune system to treat cancer, infectious diseases,allergy, asthma, autoimmune disease, and other disorders and to helpprotect against opportunistic infections following cancer chemotherapy.The strong yet balanced, cellular and humoral immune responses thatresult from, for example, TLR agonist stimulation reflect the body's ownnatural defense system against invading pathogens and cancerous cells.

Thus the agonists of nucleic acid-interacting complexes useful in someaspects of the invention as a vaccine for the treatment of a subject atrisk of developing or a subject having allergy or asthma, an infectionwith an infectious organism or a cancer in which a specific cancerantigen has been identified. The agonists of nucleic acid-interactingcomplexes can also be given without the antigen or allergen forprotection against infection, allergy or cancer, and in this caserepeated doses may allow longer term protection. A subject at risk asused herein is a subject who has any risk of exposure to an infectioncausing pathogen or a cancer or an allergen or a risk of developingcancer. For instance, a subject at risk may be a subject who is planningto travel to an area where a particular type of infectious agent isfound or it may be a subject who through lifestyle or medical proceduresis exposed to bodily fluids which may contain infectious organisms ordirectly to the organism or even any subject living in an area where aninfectious organism or an allergen has been identified. Subjects at riskof developing infection also include general populations to which amedical agency recommends vaccination with a particular infectiousorganism antigen. If the antigen is an allergen and the subject developsallergic responses to that particular antigen and the subject may beexposed to the antigen, i.e., during pollen season, then that subject isat risk of exposure to the antigen.

A subject having an infection is a subject that has been exposed to aninfectious pathogen and has acute or chronic detectable levels of thepathogen in the body. The CpG immunostimulatory oligonucleotides can beused with or without an antigen to mount an antigen specific systemic ormucosal immune response that is capable of reducing the level of oreradicating the infectious pathogen. An infectious disease, as usedherein, is a disease arising from the presence of a foreignmicroorganism in the body. It is particularly important to developeffective vaccine strategies and treatments to protect the body'smucosal surfaces, which are the primary site of pathogenic entry.

A subject having an allergy is a subject that has or is at risk ofdeveloping an allergic reaction in response to an allergen. An allergyrefers to acquired hypersensitivity to a substance (allergen). Allergicconditions include but are not limited to eczema, allergic rhinitis orcoryza, hay fever, conjunctivitis, bronchial asthma, urticaria (hives)and food allergies, and other atopic conditions.

A subject having a cancer is a subject that has detectable cancerouscells. The cancer may be a malignant or non-malignant cancer. Cancers ortumors include but are not limited to biliary tract cancer; braincancer; breast cancer; cervical cancer; choriocarcinoma; colon cancer;endometrial cancer; esophageal cancer; gastric cancer; intraepithelialneoplasms; lymphomas; liver cancer; lung cancer (e.g. small cell andnon-small cell); melanoma; neuroblastomas; oral cancer; ovarian cancer;pancreas cancer; prostate cancer; rectal cancer; sarcomas; skin cancer;testicular cancer; thyroid cancer; and renal cancer, as well as othercarcinomas and sarcomas. In one embodiment the cancer is hairy cellleukemia, chronic myelogenous leukemia, cutaneous T-cell leukemia,multiple myeloma, follicular lymphoma, malignant melanoma, squamous cellcarcinoma, renal cell carcinoma, prostate carcinoma, bladder cellcarcinoma, or colon carcinoma.

A subject shall mean a human or vertebrate animal including but notlimited to a dog, cat, horse, cow, pig, sheep, goat, turkey, chicken,primate, e.g., monkey, and fish (aquaculture species), e.g. salmon.Thus, the invention can also be used to treat cancer and tumors,infections, and allergy/asthma in non human subjects.

As used herein, the term treat, treated, or treating when used withrespect to an disorder such as an infectious disease, cancer, allergy,or asthma refers to a prophylactic treatment which increases theresistance of a subject to development of the disease (e.g., toinfection with a pathogen) or, in other words, decreases the likelihoodthat the subject will develop the disease (e.g., become infected withthe pathogen) as well as a treatment after the subject has developed thedisease in order to fight the disease (e.g., reduce or eliminate theinfection) or prevent the disease from becoming worse.

An antigen as used herein is a molecule capable of provoking an immuneresponse. Antigens include but are not limited to cells, cell extracts,proteins, polypeptides, peptides, polysaccharides, polysaccharideconjugates, peptide and non-peptide mimics of polysaccharides and othermolecules, small molecules, lipids, glycolipids, carbohydrates, virusesand viral extracts and muticellular organisms such as parasites andallergens. The term antigen broadly includes any type of molecule whichis recognized by a host immune system as being foreign. Antigens includebut are not limited to cancer antigens, microbial antigens, andallergens.

As used herein, the terms “cancer antigen” and “tumor antigen” are usedinterchangeably to refer to antigens which are differentially expressedby cancer cells and can thereby be exploited in order to target cancercells. Cancer antigens are antigens which can potentially stimulateapparently tumor-specific immune responses. Some of these antigens areencoded, although not necessarily expressed, by normal cells. Theseantigens can be characterized as those which are normally silent (i.e.,not expressed) in normal cells, those that are expressed only at certainstages of differentiation and those that are temporally expressed suchas embryonic and fetal antigens. Other cancer antigens are encoded bymutant cellular genes, such as oncogenes (e.g., activated ras oncogene),suppressor genes (e.g., mutant p53), fusion proteins resulting frominternal deletions or chromosomal translocations. Still other cancerantigens can be encoded by viral genes such as those carried on RNA andDNA tumor viruses. A cancer antigen is a compound, such as a peptide orprotein, associated with a tumor or cancer cell surface and which iscapable of provoking an immune response when expressed on the surface ofan antigen presenting cell in the context of an MHC molecule. Cancerantigens can be prepared from cancer cells either by preparing crudeextracts of cancer cells, for example, as described in Cohen, et al.,1994, Cancer Research, 54:1055, by partially purifying the antigens, byrecombinant technology, or by de novo synthesis of known antigens.

A microbial antigen as used herein is an antigen of a microorganism andincludes but is not limited to virus, bacteria, parasites, and fungi.Such antigens include the intact microorganism as well as naturalisolates and fragments or derivatives thereof and also syntheticcompounds which are identical to or similar to natural microorganismantigens and induce an immune response specific for that microorganism.A compound is similar to a natural microorganism antigen if it inducesan immune response (humoral and/or cellular) to a natural microorganismantigen. Such antigens are used routinely in the art and are well knownto those of ordinary skill in the art.

Examples of viruses that have been found in humans include but are notlimited to: Retroviridae (e.g. human immunodeficiency viruses, such asHIV-1 (also referred to as HDTV-III, LAVE or HTLV-III/LAV, or HIV-III;and other isolates, such as HIV-LP; Picornaviridae (e.g. polio viruses,hepatitis A virus; enteroviruses, human Coxsackie viruses, rhinoviruses,echoviruses); Calciviridae (e.g. strains that cause gastroenteritis);Togaviridae (e.g. equine encephalitis viruses, rubella viruses);Flaviridae (e.g. dengue viruses, encephalitis viruses, yellow feverviruses); Coronoviridae (e.g. coronaviruses); Rhabdoviradae (e.g.vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g. ebolaviruses); Paramyxoviridae (e.g. parainfluenza viruses, mumps virus,measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g.influenza viruses); Bungaviridae (e.g. Hantaan viruses, bunga viruses,phleboviruses and Nairo viruses); Arena viridae (hemorrhagic feverviruses); Reoviridae (e.g. reoviruses, orbiviurses and rotaviruses);Birnaviridae; Hepadnaviridae (Hepatitis B virus); Parvovirida(parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses);Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus(HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpesvirus; Poxviridae (variola viruses, vaccinia viruses, pox viruses); andIridoviridae (e.g. African swine fever virus); and unclassified viruses(e.g. the agent of delta hepatitis (thought to be a defective satelliteof hepatitis B virus), the agents of non-A, non-B hepatitis (class1=internally transmitted; class 2=parenterally transmitted (i.e.Hepatitis C); Norwalk and related viruses, and astroviruses).

Both gram negative and gram positive bacteria serve as antigens invertebrate animals. Such gram positive bacteria include, but are notlimited to, Pasteurella species, Staphylococci species, andStreptococcus species. Gram negative bacteria include, but are notlimited to, Escherichia coli, Pseudomonas species, and Salmonellaspecies. Specific examples of infectious bacteria include but are notlimited to, Helicobacter pyloris, Borelia burgdorferi, Legionellapneumophilia, Mycobacteria sps (e.g. M. tuberculosis, M. avium, M.intracellulare, M. kansaii, M. gordonae), Staphylococcus aureus,Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes,Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae(Group B Streptococcus), Streptococcus (viridans group), Streptococcusfaecalis, Streptococcus bovis, Streptococcus (anaerobic sps.),Streptococcus pneumoniae, pathogenic Campylobacter sp., Enterococcussp., Haemophilus influenzae, Bacillus antracis, corynebacteriumdiphtheriae, corynebacterium sp., Erysipelothrix rhusiopathiae,Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes,Klebsiella pneumoniae, Pasturella multocida, Bacteroides sp.,Fusobacterium nucleatum, Streptobacillus moniliformis, Treponemapallidium, Treponema pertenue, Leptospira, Rickettsia, and Actinomycesisraelli.

Examples of fungi include Cryptococcus neoformans, Histoplasmacapsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydiatrachomatis, Candida albicans.

Other infectious organisms (i.e., protists) include Plasmodium spp. suchas Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, andPlasmodium vivax and Toxoplasma gondii. Blood-borne and/or tissuesparasites include Plasmodium spp., Babesia microti, Babesia divergens,Leishmania tropica, Leishmania spp., Leishmania braziliensis, Leishmaniadonovani, Trypanosoma gambiense and Trypanosoma rhodesiense (Africansleeping sickness), Trypanosoma cruzi (Chagas' disease), and Toxoplasmagondii.

Other medically relevant microorganisms have been described extensivelyin the literature, e.g., see C. G. A Thomas, Medical Microbiology,Bailliere Tindall, Great Britain 1983, the entire contents of which ishereby incorporated by reference.

An allergen refers to a substance (antigen) that can induce an allergicor asthmatic response in a susceptible subject. The list of allergens isenormous and can include pollens, insect venoms, animal dander dust,fungal spores and drugs (e.g. penicillin). Examples of natural, animaland plant allergens include but are not limited to proteins specific tothe following genuses: Canine (Canis familiaris); Dermatophagoides (e.g.Dermatophagoides farinae); Felis (Felis domesticus); Ambrosia (Ambrosiaartemiisfolia; Lolium (e.g. Lolium perenne or Lolium multiflorum);Cryptomeria (Cryptomeria japonica); Alternaria (Alternaria alternata);Alder; Alnus (Alnus gultinoasa); Betula (Betula verrucosa); Quercus(Quercus alba); Olea (Olea europa); Artemisia (Artemisia vulgaris);Plantago (e.g. Plantago lanceolata); Parietaria (e.g. Parietariaofficinalis or Parietaria judaica); Blattella (e.g. Blattellagermanica); Apis (e.g. Apis multiflorum); Cupressus (e.g. Cupressussempervirens, Cupressus arizonica and Cupressus macrocarpa); Juniperus(e.g. Juniperus sabinoides, Juniperus virginiana, Juniperus communis andJuniperus ashei); Thuya (e.g. Thuya orientalis); Chamaecyparis (e.g.Chamaecyparis obtusa); Periplaneta (e.g. Periplaneta americana);Agropyron (e.g. Agropyron repens); Secale (e.g. Secale cereale);Triticum (e.g. Triticum aestivum); Dactylis (e.g. Dactylis glomerata);Festuca (e.g. Festuca elatior); Poa (e.g. Poa pratensis or Poacompressa); Avena (e.g. Avena sativa); Holcus (e.g. Holcus lanatus);Anthoxanthum (e.g. Anthoxanthum odoratum); Arrhenatherum (e.g.Arrhenatherum elatius); Agrostis (e.g. Agrostis alba); Phleum (e.g.Phleum pratense); Phalaris (e.g. Phalaris arundinacea); Paspalum (e.g.Paspalum notatum); Sorghum (e.g. Sorghum halepensis); and Bromus (e.g.Bromus inermis).

The nanoscale constructs of the invention may also be coated with oradministered in conjunction with an anti-microbial agent. Ananti-microbial agent, as used herein, refers to a naturally-occurring orsynthetic compound which is capable of killing or inhibiting infectiousmicroorganisms. The type of anti-microbial agent useful according to theinvention will depend upon the type of microorganism with which thesubject is infected or at risk of becoming infected. Anti-microbialagents include but are not limited to anti-bacterial agents, anti-viralagents, anti-fungal agents and anti-parasitic agents. Phrases such as“anti-infective agent”, “anti-bacterial agent”, “anti-viral agent”,“anti-fungal agent”, “anti-parasitic agent” and “parasiticide” havewell-established meanings to those of ordinary skill in the art and aredefined in standard medical texts. Briefly, anti-bacterial agents killor inhibit bacteria, and include antibiotics as well as other syntheticor natural compounds having similar functions. Antibiotics are lowmolecular weight molecules which are produced as secondary metabolitesby cells, such as microorganisms. In general, antibiotics interfere withone or more bacterial functions or structures which are specific for themicroorganism and which are not present in host cells. Anti-viral agentscan be isolated from natural sources or synthesized and are useful forkilling or inhibiting viruses. Anti-fungal agents are used to treatsuperficial fungal infections as well as opportunistic and primarysystemic fungal infections. Anti-parasite agents kill or inhibitparasites.

Antibacterial agents kill or inhibit the growth or function of bacteria.A large class of antibacterial agents is antibiotics. Antibiotics, whichare effective for killing or inhibiting a wide range of bacteria, arereferred to as broad spectrum antibiotics. Other types of antibioticsare predominantly effective against the bacteria of the classgram-positive or gram-negative. These types of antibiotics are referredto as narrow spectrum antibiotics. Other antibiotics which are effectiveagainst a single organism or disease and not against other types ofbacteria, are referred to as limited spectrum antibiotics. Antibacterialagents are sometimes classified based on their primary mode of action.In general, antibacterial agents are cell wall synthesis inhibitors,cell membrane inhibitors, protein synthesis inhibitors, nucleic acidsynthesis or functional inhibitors, and competitive inhibitors.

Antiviral agents are compounds which prevent infection of cells byviruses or replication of the virus within the cell. There are manyfewer antiviral drugs than antibacterial drugs because the process ofviral replication is so closely related to DNA replication within thehost cell, that non-specific antiviral agents would often be toxic tothe host. There are several stages within the process of viral infectionwhich can be blocked or inhibited by antiviral agents. These stagesinclude, attachment of the virus to the host cell (immunoglobulin orbinding peptides), uncoating of the virus (e.g. amantadine), synthesisor translation of viral mRNA (e.g. interferon), replication of viral RNAor DNA (e.g. nucleotide analogues), maturation of new virus proteins(e.g. protease inhibitors), and budding and release of the virus.

The constructs of the invention may also be administered in conjunctionwith a therapeutic or diagnostic antibody. In one embodiment, theantibody may be selected from the group consisting of Ributaxin,Herceptin, Quadramet, Panorex, IDEC-Y2B8, BEC2, C225, Oncolym, SMARTM195, ATRAGEN, Ovarex, Bexxar, LDP-03, ior t6, MDX-210, MDX-11, MDX-22,OV103, 3622W94, anti-VEGF, Zenapax, MDX-220, MDX-447, MELIMMUNE-2,MELIMMUNE-1, CEACIDE, Pretarget, NovoMAb-G2, TNT, Gliomab-H, GNI-250,EMD-72000, LymphoCide, CMA 676, Monopharm-C, 4B5, ior egf.r3, ior c5,BABS, anti-FLK-2, MDX-260, ANA Ab, SMART 1D10 Ab, SMART ABL 364 Ab,rituxan, bevacizumab, and ImmuRAIT-CEA.

The agonists of nucleic acid-interacting complexes are also useful fortreating and preventing autoimmune disease. Autoimmune disease is aclass of diseases in which an subject's own antibodies react with hosttissue or in which immune effector T cells are autoreactive toendogenous self peptides and cause destruction of tissue. Thus an immuneresponse is mounted against a subject's own antigens, referred to asself antigens. Autoimmune diseases include but are not limited torheumatoid arthritis, Crohn's disease, multiple sclerosis, systemiclupus erythematosus (SLE), autoimmune encephalomyelitis, myastheniagravis (MG), Hashimoto's thyroiditis, Goodpasture's syndrome, pemphigus(e.g., pemphigus vulgaris), Grave's disease, autoimmune hemolyticanemia, autoimmune thrombocytopenic purpura, scleroderma withanti-collagen antibodies, mixed connective tissue disease, polymyositis,pernicious anemia, idiopathic Addison's disease, autoimmune-associatedinfertility, glomerulonephritis (e.g., crescentic glomerulonephritis,proliferative glomerulonephritis), bullous pemphigoid, Sjögren'ssyndrome, insulin resistance, and autoimmune diabetes mellitus.

A “self-antigen” as used herein refers to an antigen of a normal hosttissue. Normal host tissue does not include cancer cells. Thus an immuneresponse mounted against a self-antigen, in the context of an autoimmunedisease, is an undesirable immune response and contributes todestruction and damage of normal tissue, whereas an immune responsemounted against a cancer antigen is a desirable immune response andcontributes to the destruction of the tumor or cancer. Thus, in someaspects of the invention aimed at treating autoimmune disorders it isnot recommended that the CpG immunostimulatory nucleic acids beadministered with self antigens, particularly those that are the targetsof the autoimmune disorder.

In other instances, the CpG immunostimulatory nucleic acids may bedelivered with low doses of self-antigens. A number of animal studieshave demonstrated that mucosal administration of low doses of antigencan result in a state of immune hyporesponsiveness or “tolerance.” Theactive mechanism appears to be a cytokine-mediated immune deviation awayfrom a Thl towards a predominantly Th2 and Th3 (i.e., TGF-β dominated)response. The active suppression with low dose antigen delivery can alsosuppress an unrelated immune response (bystander suppression) which isof considerable interest in the therapy of autoimmune diseases, forexample, rheumatoid arthritis and SLE. Bystander suppression involvesthe secretion of Th1-counter-regulatory, suppressor cytokines in thelocal environment where proinflammatory and Th1 cytokines are releasedin either an antigen-specific or antigen-nonspecific manner. “Tolerance”as used herein is used to refer to this phenomenon. Indeed, oraltolerance has been effective in the treatment of a number of autoimmunediseases in animals including: experimental autoimmune encephalomyelitis(EAE), experimental autoimmune myasthenia gravis, collagen-inducedarthritis (CIA), and insulin-dependent diabetes mellitus. In thesemodels, the prevention and suppression of autoimmune disease isassociated with a shift in antigen-specific humoral and cellularresponses from a Th1 to Th2/Th3 response.

In another aspect, the present invention is directed to a kit includingone or more of the compositions previously discussed. A “kit,” as usedherein, typically defines a package or an assembly including one or moreof the compositions of the invention, and/or other compositionsassociated with the invention, for example, as previously described.Each of the compositions of the kit, if present, may be provided inliquid form (e.g., in solution), or in solid form (e.g., a driedpowder). In certain cases, some of the compositions may be constitutableor otherwise processable (e.g., to an active form), for example, by theaddition of a suitable solvent or other species, which may or may not beprovided with the kit. Examples of other compositions that may beassociated with the invention include, but are not limited to, solvents,surfactants, diluents, salts, buffers, emulsifiers, chelating agents,fillers, antioxidants, binding agents, bulking agents, preservatives,drying agents, antimicrobials, needles, syringes, packaging materials,tubes, bottles, flasks, beakers, dishes, frits, filters, rings, clamps,wraps, patches, containers, tapes, adhesives, and the like, for example,for using, administering, modifying, assembling, storing, packaging,preparing, mixing, diluting, and/or preserving the compositionscomponents for a particular use, for example, to a sample and/or asubject.

In some embodiments, a kit associated with the invention includes one ormore nanoparticle cores, such as a nanoparticle core that comprisesgold. A kit can also include one or more agonists of nucleicacid-interacting complexes. A kit can also include one or more antigens.

A kit of the invention may, in some cases, include instructions in anyform that are provided in connection with the compositions of theinvention in such a manner that one of ordinary skill in the art wouldrecognize that the instructions are to be associated with thecompositions of the invention. For instance, the instructions mayinclude instructions for the use, modification, mixing, diluting,preserving, administering, assembly, storage, packaging, and/orpreparation of the compositions and/or other compositions associatedwith the kit. In some cases, the instructions may also includeinstructions for the use of the compositions, for example, for aparticular use, e.g., to a sample. The instructions may be provided inany form recognizable by one of ordinary skill in the art as a suitablevehicle for containing such instructions, for example, written orpublished, verbal, audible (e.g., telephonic), digital, optical, visual(e.g., videotape, DVD, etc.) or electronic communications (includingInternet or web-based communications), provided in any manner.

In some embodiments, the present invention is directed to methods ofpromoting one or more embodiments of the invention as discussed herein.As used herein, “promoting” includes all methods of doing businessincluding, but not limited to, methods of selling, advertising,assigning, licensing, contracting, instructing, educating, researching,importing, exporting, negotiating, financing, loaning, trading, vending,reselling, distributing, repairing, replacing, insuring, suing,patenting, or the like that are associated with the systems, devices,apparatuses, articles, methods, compositions, kits, etc. of theinvention as discussed herein. Methods of promotion can be performed byany party including, but not limited to, personal parties, businesses(public or private), partnerships, corporations, trusts, contractual orsub-contractual agencies, educational institutions such as colleges anduniversities, research institutions, hospitals or other clinicalinstitutions, governmental agencies, etc. Promotional activities mayinclude communications of any form (e.g., written, oral, and/orelectronic communications, such as, but not limited to, e-mail,telephonic, Internet, Web-based, etc.) that are clearly associated withthe invention.

In one set of embodiments, the method of promotion may involve one ormore instructions. As used herein, “instructions” can define a componentof instructional utility (e.g., directions, guides, warnings, labels,notes, FAQs or “frequently asked questions,” etc.), and typicallyinvolve written instructions on or associated with the invention and/orwith the packaging of the invention. Instructions can also includeinstructional communications in any form (e.g., oral, electronic,audible, digital, optical, visual, etc.), provided in any manner suchthat a user will clearly recognize that the instructions are to beassociated with the invention, e.g., as discussed herein.

The present invention is further illustrated by the following Examples,which in no way should be construed as further limiting. The entirecontents of all of the references (including literature references,issued patents, published patent applications, and co pending patentapplications) cited throughout this application are hereby expresslyincorporated by reference.

EXAMPLES Example 1

Materials and Methods

Synthesis of Immunostimulatory SNAs

Synthesis of immunostimulatory SNAs (isSNA) is achieved as describedelsewhere⁷⁻¹² with the following essential modifications. In brief, 20mL 13 nm gold colloid is mixed with 10% Tween 20 and TLR agonistsequence of sulfhydryl-modified nucleic acid (TLR 3, 7/8, 9) atappropriate concentration, such as 5 uM and allowed to react overnight.Addition of antigen can be achieved similar to methods as describedelsewhere.¹⁰ Purification of SNAs can be achieved by repeatedultracentrifugation at 75,000×g for 30 min.

Cell Lines

RAW 264.7 cell lines were obtained from ATCC. RAW-Blue macrophages wereobtained from InVivoGen. Ramos-Blue and THP1-XBlue cells were obtainedfrom InVivoGen. All were cultured according to the distributorrecommendations.

Results and Discussion

It was found that the nanoscale constructs of the invention markedlyenhance potency in macrophages over unformulated agonists of nucleicacid-interacting complexes (CpG oligonucleotides) in solution (FIG. 2).RAW-Blue macrophages were plated at 65 k cells per well and allowed toadhere overnight. On the day of experiments, cells were treated withAST-008-ps or CpG 1826-ps at the indicated concentration of oligo for 30min (top panel), 4 h (middle panel), or overnight (bottom panel). For 30min and 4 h time points, the entire supernatant was aspirated, the cellswere washed, and complete growth medium without test agents wereadministered. At the overnight time point, the activation state of thecells was determined using the QuantiBlue assay kit. The results showthat particularly at short time points, AST-008-ps demonstrates asignificantly lower EC50 than CpG 1826-ps of 188 nM as compared to 17800nM (9-1000-fold reduction one standard deviation from the mean). At 4 h,AST-008-ps demonstrates a lower EC50 (32 nM vs 57 nm) and greateractivation state than CpG 1826-ps. This difference became narrowerfollowing overnight incubation, at which point the EC50s were notstatistically different from each other. This suggests that underconditions where residence time of the agent with the target cell islimited, particularly to 30 minutes or below, the AST-008-ps formulationmay result in more rapid and robust immune activation.

It was also demonstrated that the nanoscale constructs of the inventionmarkedly enhanced potency in macrophages over unformulated agonists ofnucleic acid-interacting complexes (CpG oligonucleotides) in solutionfollowing overnight incubation (FIG. 3). RAW-Blue macrophages wereplated at 65k cells per well and allowed to adhere overnight. On the dayof experiments, AST-007-po, AST-007-ps, AST-008-po, AST-008-ps, CpG1826-po, CpG 1826-ps were incubated with the cells overnight. The degreeof activation was then determined using the QuantiBlue assay kit. Theresults show that for compounds containing only phosphodiester (−po)linkages (top panel), AST-007-po and AST-008-po are ˜50-150 fold morepotent as determined by their EC50 values than CpG 1826-po (191 nM and194 nM as compared to 16032 nM, respectively). Withphosphorothioate-modified compounds (-ps, bottom panel), AST-007-ps andAST-008-ps are approximately equivalent to CpG 1826-ps, with an EC50 of29 nM, 27 nM, and 20 nM, respectively.

Levels of cytokine secretion following exposure to the nanoscaleconstructs of the invention versus unformulated agonists of nucleicacid-interacting complexes (CpG oligonucleotides) in solution wasexamined. The effect on cytokine induction was examined for botholigonucleotides having phosphodiester and phosphorothioateinternucleotide linkages in both the nanoparticle and the TRL agonistgroups (FIG. 4). RAW-Blue macrophages were plated at 65k cells per welland allowed to adhere overnight. On the day of experiments, theindicated compounds were incubated with the cells overnight. The degreeof cytokine secretion was determined by collecting the supernatant andmeasuring the concentration of the indicated cytokines by ELISA(TNF-alpha- top panel, IL-12- bottom right panel, IL-6- bottom leftpanel). The results show significantly higher cytokine secretion atlower doses are possible using AST-008-ps and AST-008-po than CpG1826-ps, CpG 1826-po, and the indicated controls. For example, toachieve greater than 2000 pg/mL TNF-alpha, less than 100 nM AST-008-pswas needed and less than 1000 nM AST-007-po was needed, but greater than1000 nM CpG 1826-ps was required.

Next, TLR9 activation in response to stimulation with a nanoscale of theinvention having a phosphodiester CpG oligonucleotide in comparison withphosphodiester and phosphorothioate CpG oligonucleotides in solution wasexamined (FIG. 5). Ramos-Blue or THP1-XBlue cells were seeded andactivated according to the manufacturer's recommended protocol using theindicated compounds and controls. Remarkably, AST-007-po, AST-008-po,and AST-009-po demonstrate comparable activation at similar doses thanCpG 7909-ps, a known and optimized TLR 9 agonist. In addition,activation appeared to be dependent on TLR 9, as the TLR 9 agonistinsensitive THP1-XBlue cells did not demonstrate any activation.

It was determined that a nanoscale construct of the invention had amultiple fold increase in potency of over several different CpG oligosequences (FIG. 6). Ramos-Blue cells were seeded and activated accordingto the manufacturer's recommended protocol using the indicated compoundsand controls. Oligo 1826 (top panel) and 1668 (bottom panel) weretested. Notably, SNA compounds demonstrate significantly lower EC50values than free oligos, independent of chemistry, as compared tocontrols. This suggests that for these sequences, the SNA formulation ofoligos is several fold more potent.

The effects of modulating nanoparticle core size was examined (FIGS.7A-7B). Raw Blue cells were plated and treated with the indicatedagonists with different gold core sizes, ranging from 3.5 nm to 13 nmusing the indicated oligos. The results show that smaller gold coresizes appear to demonstrate the potential to enhance agonist activity invitro.

The nanoscale constructs of the invention were observed to have a morerapid and sustained activation than CpG oligo (FIG. 8). Cells wereplated as described and activation was measured using QuantiBlue. Theresults show that at 6 nM oligo, the PS SNAs demonstrate significantlymore activation than free 1668 PS oligos.

The ability of phosphorothioate modifications to modulate agonistactivity in a sequence-dependent manner was examined (FIG. 9). Raw Bluecells were plated and treated with the indicated agonists. Oligo 1826(top panel) and 1668 (bottom panel) were tested. The results show thatinternal phosphorothioate modifications (C*G) and two 5′phosphorothioate linkages (5′PS2) have an effect on the activity ofimmunostimulatory SNAs that appears to be sequence dependent.

The ability of oligonucleotide loading density to affect agonistactivity was assessed (FIG. 10). V2 indicates that the construct wascompletely gold coated prior to oligo addition to the gold core.Phosphodiester oligonucleotides (top panel) and phosphorothioateoligonucleotides (bottom panel) were tested. The data shows that thedensity of oligonucleotide on the surface of the gold will modulate theactivity of the immunostimulatory construct.

A time course of activation of CpG PO/PO nanoscale constructs wasstudied (FIG. 11). The tested constructs are not activated until >4 hrof incubation. Raw Blue cells were plated and treated with the indicatedagonists. The results show that PO and PO SNAs do not robustly activateadherent RAW Blue cells until greater than 4 hours of incubation.

5′Chol CpG PO nanoscale constructs showed activation in low nM range,while 5′C18 abrogated the activity (FIG. 12). A 5′ cholesterolmodification (5′Chol) may increase the potency of the agonist,particularly at low concentrations in a oligo-dose-independent manner.Modification of the 5′ end with a C18 molecule (5′C18) appears toeliminate the activity altogether.

Pre-plated macrophages are more primed for subsequent activation (FIG.13). RAW Blue macrophages that were plated overnight prior to additionof the agonist compounds (top) generally demonstrate greater activationthan when the cells are plated at the same time as the agonist compoundis added (bottom).

It was demonstrated that low levels of IFN-gamma secretion bymacrophages (FIG. 14). RAW-Blue macrophages were plated at 65 k cellsper well and allowed to adhere overnight. On the day of experiments, theindicated compounds were incubated with the cells overnight. The degreeof cytokine secretion (either 24 hours—left panel or 48 hours—rightpanel after treatment) was determined by collecting the supernatant andmeasuring the concentration of the indicated cytokines by ELISA. Theresults show that IFN-gamma is not produced to an appreciable extent byRAW Blue macrophages stimulated with these compounds.

Example 2 Immuno-Oncology and Immunotherapies

Immunotherapeutic SNAs (i.e., AST-008) provide a novel and versatiletechnology platform. Their multi-valent immunomodulator deliveryoptimizes responses, while profound tumor reduction in a lymphoma modelhas been observed. Additionally, they trigger a potent and balanced Tcell response in vivo greater than that of free oligonucleotide or alum.SNAs can co-present therapeutic vaccine antigen and adjuvant on a singlenanoparticle and have enhanced activity and faster kinetics than freeimmunostimulatory CpG oligodeoxynucleotides. Furthermore, SNAs have thepotential to simultaneously target multiple immunostimulatory receptors(e.g. TLR 3, 4, 7/8, 9). They can be used, for example, in cancerimmunotherapy and vaccines (prophylactic or therapeutic).

A schematic of an immunotherapeutic SNA (AST-008) is shown in FIG. 15.The SNAs can co-present a therapeutic vaccine antigen and adjuvant on asingle nanoparticle, and may simultaneous target multipleimmunostimulatory receptors (e.g. TLR 3, 4, 7/8, 9).

A schematic demonstrating how AST-008 can enter endosomes via triggeredendocytosis is shown in FIG. 16. AST-008 once in the endosomes can beused for versatile immune system stimulation. Within the endosome,AST-008 stimulates immune system signaling via the TLR 9 receptor, amolecular target for SNA therapy, leading to both innate and adaptiveimmune responses. AST-008 may also target TLR 3, 4, 7/8, resulting ininnate and adaptive immune responses.

AST-008 induces higher pro-inflammatory responses than corresponding CpGoligodeoxynucleotides (oligo) in vitro. An assay demonstrating thisfinding was conducted. The data is shown as a set of graphs in FIGS. 17Aand 17B. FIG. 17A shows the expression levels of TNF, IL-12, and IL-6induced by CTL oligo, CTL SNA, CpG 1826, and AST-008. FIG. 17B presentsthe NF-κB activation stemming from the indicated agents.

AST-008 also targets draining lymph nodes after administration of asingle subcutaneous dose. AST-008 was silver-stained to enhance lightscattering of the gold core, and then counterstained with eosin. 4×bright field magnification was used. The data is shown in FIG. 18.

The structures of the invention are useful for stimulating a robustimmune response in vivo. For instance, FIG. 19 is a graph illustratingthe in vivo activity of AST-008. Mice were given a 50 μL bolus tail vein(intravenous) injection of 5.1 nmol solution (AST-008-po, AST-008-ps,CpG 1826-po, CpG 1826-ps, GpC-po SNA, GpC-ps SNA, GpC-po, or GpC-ps) andthen analyzed for IL-12 expression 1, 3, and 6 hours after injection (24mice per group, 3 per each time point). IL-12 levels are expressed asthe fold over PBS. AST-008 architecture enhances the induction of IL-12by approximately 20-fold over free oligodeoxynucleotides, and the effectwas sustained for over six hours after the initial administration. FIGS.20A-20C consist of a pair of graphs and a chart that demonstrate thatAST-008 induces both a balanced Th1/Th2 response (FIG. 20A) and a higherIgG2a antibody (FIG. 20B) response than alum or CpG oligonucleotides.The results are tabulated in FIG. 20C. **p<0.01. FIGS. 21A-21B show thatAST-008 induces cellular responses more effectively than alum or CpGoligonucleotides. FIG. 21A schematically represents the protocol:splenocytes were grown for 28 days, challenged on Day 0 and Day 21, andthen restimulated with SIINFEKL and probed for INF-γ with ELISPOT on Day28. FIG. 21B is a graph depicting the results. ****p<0.0001.

The structures have been shown to produce a dramatic anti-tumor responsein vivo as well. FIGS. 22A-22B demonstrate that AST-008 induces aprofound tumor-clearing immune response in an in vivo lymphoma model.FIG. 22A illustrates the protocol: the right flanks of C57BL/6 mice wereinjected with 1×10⁶ E.G7-OVA lymphoma (11 per group). The mice were thenchallenged three times with 100 μg OVA s.c., 1.8 μg OVA₂₅₇₋₂₆₄ s.c., and0.92 nmol oligo in AST-008, and sacrificed at 2000 mm³. FIG. 13 is agraph of the results. *p<0.05 using Two-way ANOVA. FIGS. 23A-23B showthat AST-008 exhibits superior anti-tumor activity and longer survivalthan CpG oligodeoxynucleotides. The graphs show the tumor volume (FIG.23A) and percent survival (FIG. 23B) after C57BL/6 mice were injectedwith 1×10⁶ E.G7-OVA lymphoma in their right flanks (11 per group) andthen were challenged three times with PBS, PBS and OVA, CpG 1826 andOVA, or AST-008 and OVA. *p<0.05.

TABLE 1 Key to symbols SEQ ID Name Oligo Sequence (5′-3′) FormulationNO: AST- TCCATGACGTTCCTGATGCT/ Conjugated to 13 36 007-po,iSp18//iSp18//iSp18// nm gold core via CpG 3ThioMC3-D/ thio-gold bond1668 PO SNA AST- TCCATGACGTTCCTGACGTT/ Conjugated to 13 37 008-po,iSp18//iSp18// nm gold core via CpG 3ThioMC3-D/ thio-gold bond 1826 POSNA AST- TCGTCGTTTTGTCGTTTTGTCG Conjugated to 13 38 009-po,TT/iSp18//iSp18// nm gold core via CpG 3ThioMC3-D/ thio-gold bond7909 PO SNA AST- tccatgacgttcctgatgct/ Conjugated to 13 39 007-ps,iSp18//iSp18//iSp18// nm gold core via CpG 3ThioMC3-D/ thio-gold bond,1668 PS backfilled with SNA tetraethylene glycol AST-tccatgacgttcctgacgtt/ Conjugated to 13 40 008-ps, iSp18//iSp18//iSp18//nm gold core via CpG 3ThioMC3-D/ thio-gold bond, 1826 PS backfilled withSNA tetraethylene glycol AST- tcgtcgttttgtcgttttgtcg Conjugated to 13 41009-ps, tt/iSp18//iSp18// nm gold core via CpG 3ThioMC3-D/thio-gold bond, 7909 PS backfilled with SNA tetraethylene glycol CpGTCCATGACGTTCCTGACGTT Free 42 1826-po CpG Tccatgacgttcctgacgtt Free 431826-ps CpG TCCATGACGTTCCTGATGCT Free 44 1668-po CpGTccatgacgttcctgatgct Free 45 1668-ps CpG TCGTCGTTTTGTCGTTTTGTCG Free 467909-po TT CpG Tcgtcgttttgtcgttttgtcg Free 47 7909-ps tt rplV-poGCTTTCTTGTTGGTGTAGGTC Free 48 rplV-ps Gctttcttgttggtgtaggtc Free 49Ctrl- Sequence containing Conjugated to 13 SNA-po, no CpG motifs, allnm gold core via rplV phosphodiester thio-gold bond SNA PO linkagesCtrl- Sequence containing Conjugated to 13 SNA-ps, no CpG motifs, allnm gold core via rplV phosphorothioate thio-gold bond SNA PS linkagesLowercase indicates phosphorothioate linkages Capital indicatesphosphodiester linkages Prefix “Ctrl” indicates oligo used with no CpGmotifs present /iSp18/ internal spacer-18 (hexaethylene glycol)/3ThioMC3-D/ terminal sulfhydryl group

REFERENCES

1. Koff WC et al. Science 340:1232910-1 (2013)

2. Cluff CW. Monophosphoryl Lipid A (MPL) as an Aduvant for Anti-CancerVaccines: Clinical Results. Lipid A in Cancer Therapy, Jeannin J Ed.Landes Bioscience (2000)

3. Krieg AM. Proc Am Thorac Soc 4:289 (2007)

4. Schmidt C. Nat Biotechnol 25:825 (2007)

5. Ellis RD et al. PLOS One 10:e46094 (2012)

6. Garcon N et al. Expert Rev. Vaccines 6:723 (2007)

7. Rosi NL et al. Science 312:1027 (2006)

8. Lytton-Jean AK et al. JACS 127:12754 (2005)

9. Hurst SJ et al. Anal. Chem. 78:8313 (2006)

10. Patel PC et al. PNAS 105:17222 (2008)

11. Seferos DS et al. Nano Lett 9:308 (2009)

12. Giljohann et al. Angew. Chem. Int. Ed. 49:3280 (2010)

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

All references, including patent documents, disclosed herein areincorporated by reference in their entirety.

What is claimed is:
 1. A nanoscale construct comprising: a coronacomprised of an exterior shell composed of nucleic acid moleculesarranged in a geometrical position and forming a spherical shape arounda nanoparticle core, wherein the nucleic acid molecules are CpGoligonucleotides, wherein the nanoscale construct is about 1 nm to about40 nm in diameter, and wherein the nanoparticle core at the center ofthe corona is not metallic.
 2. The nanoscale construct of claim 1,wherein the surface density of the nucleic acid molecules is at least0.3 pmol/cm².
 3. The nanoscale construct of claim 1, wherein the nucleicacid molecules comprise a spacer.
 4. The nanoscale construct of claim 3,wherein the spacer consists of oligoethylene.
 5. The nanoscale constructof claim 1, wherein the nanoparticle core at the center of the corona ishollow.
 6. The nanoscale construct of claim 1, wherein the nanoscaleconstruct is degradable.
 7. The nanoscale construct of claim 1, whereinthe CpG oligonucleotide has a modified backbone.
 8. The nanoscaleconstruct of claim 1, further comprising an antigen.
 9. A method fordelivering a therapeutic agent to a cell comprising delivering thenanoscale construct of claim 1 to the cell.
 10. A method of treating asubject, comprising administering to the subject a nanoscale constructin an effective amount to stimulate an immune response, wherein thenanoscale construct comprises a corona having an exterior shell composedof nucleic acid molecules arranged in a geometrical position and forminga spherical shape around a nanoparticle core, wherein the nucleic acidmolecules are CpG oligonucleotides, and wherein the nanoscale constructis about 1 nm to about 40 nm in [[mean]] diameter, and wherein thenanoparticle core at the center of the corona is not metallic.
 11. Themethod of claim 10, wherein the subject has an infectious disease. 12.The method of claim 10, wherein the subject has cancer.
 13. A method foractivating a toll-like receptor (TLR), the method comprising deliveringto a cell a nanoscale construct, wherein the nanoscale constructcomprises a corona having an exterior shell composed of nucleic acidmolecules arranged in a geometrical position and forming a sphericalshape around a nanoparticle core, wherein the nucleic acid molecules areCpG oligonucleotides, wherein the nanoscale construct is about 1 nm toabout 40 nm in diameter, and wherein the nanoparticle core at the centerof the corona is not metallic.
 14. The method of claim 13, wherein thecell is in vivo.
 15. The method of claim 14, wherein the nanoscaleconstruct is administered to a subject having cancer.
 16. The nanoscaleconstruct of claim 4, wherein the oligoethylene is hexaethylene glycol.17. The nanoscale construct of claim 4, wherein the spacer consists oftwo or three hexaethylene glycols.
 18. The nanoscale construct of claim3, wherein the spacer does not comprise an oligonucleotide. (New) Thenanoscale construct of claim 1, wherein the CpG oligonucleotide has aphosphorothioate (PS) backbone.
 20. A nucleic acid molecule comprising aCpG oligonucleotide having the nucleotide sequence of SEQ ID NO: 30,wherein the nucleic acid molecule comprises a spacer consisting ofoligoethylene.
 21. The nucleic acid molecule of claim 20, wherein theCpG oligonucleotide has a phosphorothioate (PS) backbone.
 22. Thenucleic acid molecule of claim 20, wherein the oligoethylene ishexaethylene glycol.
 23. The nucleic acid molecule of claim 20, whereinthe spacer consists of two or three hexaethylene glycols.
 24. Aplurality of nanoscale constructs, each nanoscale construct comprising:a corona comprised of an exterior shell composed of nucleic acidmolecules arranged in a geometrical position and forming a sphericalshape around a nanoparticle core, wherein the nucleic acid molecules areCpG oligonucleotides, wherein the nanoscale constructs have a meandiameter of about 1 nm to about 40 nm, and wherein the nanoparticle coreat the center of the corona is not metallic.